Serotype-specific and age-dependent generation of pneumococcal polysaccharide-specific memory B-cell and antibody responses to immunization with a pneumococcal conjugate vaccine

Abstract

Glycoconjugate vaccines have dramatically reduced the incidence of encapsulated bacterial diseases in toddlers under 2 years of age, but vaccine-induced antibody levels in this age group wane rapidly. We immunized adults and 12-month-old toddlers with heptavalent pneumococcal conjugate vaccine to determine differences in B-cell and antibody responses. The adults and 12-month-old toddlers received a pneumococcal conjugate vaccine. The toddlers received a second dose at 14 months of age. The frequencies of diphtheria toxoid and serotype 4, 14, and 23F polysaccharide-specific plasma cells and memory B cells were determined by enzyme-linked immunospot assay. The toddlers had no preexisting polysaccharide-specific memory B cells or serum immunoglobulin G (IgG) antibody but had good diphtheria toxoid-specific memory responses. The frequencies of plasma cells and memory B cells increased by day 7 (P < 0.0001) in the adults and the toddlers following a single dose of conjugate, but the polysaccharide responses were significantly lower in the toddlers than in the adults (P = 0.009 to <0.001). IgM dominated the toddler antibody responses, and class switching to the IgG was serotype dependent. A second dose of vaccine enhanced the antibody and memory B-cell responses in the toddlers but not the ex vivo plasma cell responses. Two doses of pneumococcal conjugate vaccine are required in toddlers to generate memory B-cell frequencies and antibody class switching for each pneumococcal polysaccharide equivalent to that seen in adults.

FIG. 1.

Frequency of spontaneously secreting IgG AFCs (a to e) and memory B-cell-derived IgG AFCs (f to j) isolated from the peripheral blood of 12-month-old toddlers prior to and 1 week following a primary and secondary dose of Pnc7, as determined by ELISPOT assay. Immunizations were given on day 0 (d0) and day 56 (black arrows), with blood draws performed on days 0, 6 or 7, 56, and 62 or 63. IgG AFCs specific for the control antigen (tetanus toxoid) and the vaccine antigens (diphtheria toxoid and polysaccharide serotypes 4, 14, and 23F) were enumerated. The data represent the numbers of IgG AFCs/10⁶ cultured PBMCs, and the bars represent the medians.

FIG. 2.

Frequency of memory B-cell-derived IgG AFCs isolated from the peripheral blood of adults (triangles) and 12-month-old toddlers (circles). In vitro-stimulated PBMCs were isolated at day 0 for the baseline memory B-cell-derived IgG AFC frequency (a) and 1 week after a single dose of Pnc7 (b). IgG AFCs specific for the control antigen (tetanus toxoid [tet]) and the vaccine antigens (diphtheria toxoid [dip] and polysaccharide serotypes 4, 14, and 23F) were enumerated. The data represent the numbers of IgG AFCs/10⁶ cultured PBMCs, and the bars represent the medians.

FIG. 3.

Comparison of in vitro-stimulated memory B-cell derived IgG AFC frequency in adults (triangles) and 12-month-old toddlers (circles). The responses to the single dose of Pnc7 in the adults and the toddlers at day 28 were compared to those at day 56 (a). The frequency of memory IgG AFCs achieved in the adults 1 week after one Pnc7 dose was compared with that achieved by the toddlers 1 week after a second Pnc7 dose (b). IgG AFCs specific for the control antigen (tetanus toxoid [tet]) and the vaccine antigens (diphtheria toxoid [dip] and polysaccharide serotypes 4, 14, and 23F) were enumerated. The data represent the numbers of IgG AFCs/10⁶ cultured PBMCs, and the bars represent the medians.

FIG. 4.

Frequency of spontaneously secreting IgG AFCs in adults after a single dose of Pnc7 (triangles) and 12-month-old toddlers after a single dose (closed circles) and a second dose (open circles) of Pnc7. PBMCs were isolated prior to and 1 week after each dose of vaccine and were allowed to spontaneously secrete IgG onto ELISPOT assay plate wells coated with the control antigen (tetanus toxoid [tet]) and the vaccine antigens (diphtheria toxoid [dip] and polysaccharide serotypes 4, 14, and 23F). The spots were enumerated and expressed as the numbers of IgG AFCs/10⁶ PBMCs, and the bars represent the medians (***, P ⩽ 0.0001).

FIG. 5.

Quantification of preimmunization, antipneumococcal polysaccharide serum IgG (a), IgA (b), and IgM (c) antibody levels in adults (n = 20; triangles) and 12-month-old toddlers (n = 23; circles). Antibody levels against pneumococcal serotypes 4, 14, and 23F were quantified by ELISA. The data are plotted as the log of the individual antibody concentrations (in μg/ml), and the bars represent the GMCs.

FIG. 6.

Quantification of postimmunization, antipneumococcal polysaccharide serum IgG (a), IgA (b), and IgM (c) antibody levels. The adults received a single dose of Pnc7 (n = 20; triangles), and the 12-month-old toddlers received a single dose (n = 23; closed circles), followed by a second dose (n = 23; open circles) 2 months later. The levels of antibodies against pneumococcal serotypes 4, 14, and 23F were quantified by ELISA. The data are plotted as the log of the individual antibody concentrations (in μg/ml), and the bars represent the GMCs.